Production and characterization of clinical grade Escherichia coli derived Plasmodium falciparum 42 kDa merozoite surface protein 1 (MSP1(42)) in the absence of an affinity tag

The 42 kDa cleavage product from the carboxyl end of the Plasmodium falciparum merozoite surface protein 1 (MSP1(42)) is an important blood-stage malaria vaccine target. Several recombinant protein expression systems have been used for production of MSP1(42) including yeast (Saccharomyces cerevisiae and Pichia pastoris), Escherichia coli, baculovirus and transgenic animals. To date, all of the reported recombinant proteins include a 6 x His affinity tag to facilitate purification, including three MSP1(42) clinical grade proteins currently in human trials. Under some circumstances, the presence of the 6 x His tag may not be desirable. Therefore, we were interested to produce clinical grade MSP1(42) without a 6 x His affinity tag from E. coli inclusion bodies. We produced a recombinant MSP1(42) with a P. falciparum FUP (Uganda-Palo Alto) phenotype which accounts for a substantial proportion of the MSP1(42) protein observed in African isolates. EcMSP1(42)-FUP was produced in E. coli inclusion bodies by high cell mass induction with IPTG using 5 L and 60 L bioreactors. Isolated inclusion bodies were solubilized in 8M guanidine-HCl and the EcMSP1(42)-FUP protein refolded by rapid dilution. Refolded EcMSP1(42)-FUP was purified using hydrophobic interaction chromatography, anion exchange chromatography, and size exclusion chromatography, and subject to biochemical characterization for integrity, identity, and purity. Endotoxin and host cell protein levels were within acceptable limits for human use. The process was successfully transferred to pilot-scale production in a cGMP environment. A final recovery of 87.8 mg of clinical-grade material per liter of fermentation broth was achieved. The EcMSP1(42)-FUP clinical antigen is available for preclinical evaluation and human studies.     

猪SsBHMT基因的原核表达及条件优化

甜菜碱高半胱氨酸甲基转移酶（BHMT）是一种甲基代谢酶,催化甜菜碱转甲基到高半胱氨酸生成甲硫氨酸。对含有pET30a-SsBHMT重组质粒的菌株进行诱导表达分析,通过对菌株、IPTG诱导浓度、时间等诱导表达条件进行优化,结果表明：该目的蛋白主要以包涵体的形式出现;经过条件优化,目的蛋白表达量在上清和沉淀中均有所增加,但仍主要以包涵体的形式存在;大肠杆菌菌株ER2566为适合目的蛋白表达的菌株;在37℃,IPTG浓度为0.4 mmol/L,诱导时间为10 h的条件下,目的蛋白表达量最多。研究结果为进一步蛋白纯化与抗体制备奠定了基础。     

Galactose can be an inducer for production of therapeutic proteins by auto-induction using E. coli BL21 strains

Recently lactose mediated auto-induction in Escherichia coli has gained a lot of interest because higher protein titer could be achieved without the need to monitor growth and add inducer at the proper time. In this study a high level therapeutic protein production by auto-induction was observed in E. coli BL21 using either T7 or tac promoters in the modified Luria Bertani (mLB) medium containing soy peptone instead of tryptone in Luria Bertani (LB) medium. Based on medium analysis and spiking experiments it was found that 0.4 mM galactose from the soy peptone caused the auto-induction. E. coli cultures induced by galactose can saturate at considerably higher density than cultures induced by IPTG. Galactose is not consumed by E. coli BL21. Finally it has been demonstrated that auto-induction can be effectively used in fed-batch fermentation for the industrial production of a therapeutic protein. The principle of galactose mediated auto-induction should be able to apply to high throughput microplates, shake flasks and fed-batch fermentors for clone screening and therapeutic protein expression in E. coli gal(-) strains such as most commonly used BL21.     

Effective expression of fragments of a botulinum neurotoxin type A gene, coding for the L-chain and H-chain in E. coli, with formation of products causing protective immunity to administration of the toxin

Native Clostridium botulinum gene coding for type A neurotoxin has been used to construct recombinant derivatives coding separately for L and H polypeptide chains of the toxin. The gene derivatives have been cloned into an expression vector pET28b in E. coli BL21 (DE3) cells. The recombinant L and H proteins seem to be the major individual proteins after IPTG induction of the recombinant cells. Each of the proteins has been accumulated only in inclusion bodies. The recombinant L chain (but not H chain) has been successfully resolubilized. Each of the proteins contains six His residues on the N terminus which allows purification on Ni-agarose columns with high yield. No toxic effect has been observed for both L and H chains after injection of 10 micrograms of recombinant preparations purified from inclusion bodies. Moreover, the injection resulted in an increase in the titer of specific antibodies which protected mice from 1 DLM of type A native botulinum neurotoxin. Hence, the recombinant neurotoxin protein derivatives which are present in E. coli inclusion bodies can be a source of material for producing diagnostic and therapeutic sera against type A botulinum neurotoxin.     

Antihypertensive activity of recombinant peptide IYPR expressed in Escherichia coli as inclusion bodies

To produce more angiotensin converting enzyme inhibitory peptides (ACEIP), we have established a high-efficiency Escherichia coli expression system. The DNA-coding sequence for the recombinant protein, which was subcloned into the vector pET-30a(+), has been expressed as inclusion bodies in E. coli BL21 (DE3). The influences of induction time and concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) on the expression of recombinant protein were studied. The resulting expression level of the protein accounted for about 31% of cellular protein at a temperature of 37°C, IPTG concentration of 0.6mM and induction time of 7h. The inclusion bodies were washed, separated from the cells, and solubilized with urea. After purification by affinity chromatography, the recombinant protein was recovered with a high purity of about 90%. Molecular weight of the recombinant protein was measured using Tricine-SDS-PAGE. Peptide IYPR was obtained by cleavage of the recombinant protein with trypsin and the IC(50) value was 61 mg/L. The antihypertensive activity in spontaneously hypertensive rats (SHRs) was also investigated. Single oral administration of this peptide in 10-week old SHRs resulted in a significant reduction of systolic blood pressure to 50 mm Hg at 4 h. The data obtained provide a good reference for further development of peptide Ile-Tyr-Pro-Arg into an effective antihypertensive agent for prevention and treatment of hypertension.     

非洲猪瘟病毒CP204L基因的原核表达与单抗制备

由非洲猪瘟病毒(ASFV)引起的非洲猪瘟(ASF)给我国养猪业带来了不可估量的经济损失,严重阻碍了我国养猪业的发展,研发ASFV快速诊断试剂是目前最重要的内容之一。CP204L基因编码ASFV结构蛋白p30。本研究以克隆ASFV的CP204L基因为基础,通过基因重组技术,加入His标签,将构建的重组质粒命名为pET-28a-CP204L。将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞,37℃经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达6h,表达蛋白进行SDS-PAGE鉴定和Western Blot检测。重组蛋白纯化后免疫小鼠制备筛选单克隆抗体,Western Blot和IFA验证单抗的结合特异性。结果表明,重组的pET-28a-CP204L诱导后表达蛋白为30kD,以不可溶性包涵体形式存在;表达蛋白利用His标签进行纯化,获得纯化蛋白2mg,单克隆抗体筛选获得5株IgG亚型的ASFV p30蛋白的单抗,且均具有良好的结合活性。本研究为发展ASFV检测方法提供了基础。     

重组鲁西黄牛α干扰素融合蛋白的表达及其抗病毒活性研究

通过PCR从鲁西黄牛(Yellowcattle)基因组DNA中克隆了α干扰素(BoIFN-α)基因,并插入到pET32a 中,构建成重组原核表达质粒pET32a /BoIFN-α,进行测序和诱导表达。测序结果表明,鲁西黄牛IFN-α基因全长498个核苷酸,含一个开放阅读框(ORF),编码166个氨基酸的成熟蛋白,与已报道的牛α干扰素C亚型氨基酸组成同源性为97.6%。表达产物经SDS-PAGE分析,表达出40kD的融合蛋白,表达量占菌体总蛋白的26.7%。表达产物经镍离子螯合次氨基三乙酸(Ni-NTA)亲和层析纯化,纯化产物进行复性后在MDBK/VSV上的活性为5×105u/mg。重组牛IFN-α(rBoIFN-α)对牛轮状病毒(BRV)有一定的抑制作用,抗BRV病毒活性为1.5×105u/mg。结果显示从鲁西黄牛中克隆了IFN-α基因的一种新亚型,即BoIFN-αC2,并实现了高效表达,获得了具有较高抗病毒活性的重组干扰素产物,为重组牛干扰素的开发奠定了基础。     

Basophil Activation Test Using Recombinant Allergens: Highly Specific Diagnostic Method Complementing Routine Tests in Wasp Venom Allergy

Background

Skin testing can expose allergic subjects to potential systemic reactions, sensitization against unrelated proteins, and increased risk of future sting reactions. Therefore the continuous improvement of in vitro diagnostic methods is desirable. Recombinant allergens have been shown to improve the sensitivity of specific IgE (sIgE) detection in vitro whilst no data is available regarding their application and reliability in basophil activation test (BAT). Here we aimed to compare the specificity and sensitivity of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 in both specific IgE (sIgE) detection in vitro and basophil activation test.

Methods

sIgE detection by ELISA or ImmunoCAP and BAT towards the panel of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 were performed in 43 wasp venom allergic patients with a history of anaphylactic reaction and sIgE seropositivity, as well as 17 controls defined as subjects with a history of repetitive wasp stings but absence of any allergic symptom.

Results

The BAT performed with the recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 markedly improved the specificity of diagnosis in wasp venom allergic subjects when compared to the respective sIgE detection in serum.

Conclusions

BAT performed with the recombinant allergens Ves v 5, Ves v 3 and Ves v 1 provides an emerging highly specific in vitro method for the detection of wasp venom allergy, compared to the sIgE detection. Recombinant allergens applied to BAT represent a step forward in developing reliable in vitro tests for specific diagnosis of allergy.     

幽门螺杆菌致病岛CagL重组抗原的可溶性表达及其多克隆抗体的制备和分析*

目的:利用原核表达和蛋白质纯化技术获得高纯度的幽门螺杆菌致病岛CagL重组抗原(rCagL),利用其制备anti-CagL多克隆抗体,并分析抗体的特异性。方法:通过生物信息学软件分析rCagL的抗原结构;利用PCR长片段DNA合成技术合成不含有信号肽序列的幽门螺杆菌致病岛CagL基因,将其插入表达质粒pCzn1中,构建重组质粒pCzn1-rCagL。然后,将pCzn1-rCagL转入大肠杆菌Arctic Express中,经IPTG诱导表达后,通过Ni-IDA镍离子亲和层析纯化重组抗原rCagL,利用Western blot鉴定rCagL与His标签抗体和Anti-H. pylori抗体的免疫反应性;最后,通过rCagL辅以弗氏佐剂免疫BALB/c小鼠,制备anti-CagL多克隆抗血清,通过ELISA方法分析抗血清的特异性。结果:生物信息学软件表明重组抗原rCagL具有较好的抗原性质;重组质粒pCzn1-rCagL经双酶切和基因测序等技术鉴定,证实rCagL核苷酸序列与理论序列完全一致;基因工程菌株pCzn1-rCagL/Arctic Express在低温11℃条件经IPTG诱导表达。 SDS-PAGE实验结果证实:rCagL可实现相对高效地可溶性蛋白表达,可溶性蛋白约占包涵体的62.07%。经Ni-IDA亲和层析柱纯化,可获得高纯度rCagL,纯度约为96.6%。Western blot结果证实:重组抗原rCagL可特异性与His标签抗体和Anti-H. pylori抗体结合。ELISA结果证实:经rCagL免疫小鼠制备的多克隆抗体anti-CagL可特异性识别rCagL和H. pylori裂解物,具有较高的抗体特异性。结论:重组抗原rCagL在低温条件下可实现可溶性表达,经纯化可获得高纯度抗原蛋白;rCagL具有较好的抗原性,制备的多克隆抗体具有较好的免疫特异性,为发展H. pylori相关诊断试剂奠定了实验基础。     

精氨酸脱亚氨酶的表达、纯化和活性研究

目的：建立精氨酸脱亚氨酶的高效表达菌种和纯化工艺路线。方法：人工合成编码支原体精氨酸脱亚氨酶（arginine deiminase, ADI）的基因,构建pBV220-ADI原核表达载体,转染大肠杆菌DH5α中并诱导表达目的蛋白,离子交换层析和分子筛层析法纯化目标蛋白。采用体外精氨酸降解试验测定纯化产物活性。结果：成功构建了原核表达载体pBV220-ADI,基因侧序正确。转化大肠杆菌DH5α后筛选到高水平表达目的蛋白的菌株,目标蛋白以包含体形式存在于胞浆内,表达水平超过全菌体蛋白的35%。采用盐酸胍溶解包含体、低温条件下稀释和透析的方法进行复性。顺次采用阳离子交换和凝胶过滤层析对复性液进行纯化,最终获得纯度达到95%的活性产物。活性测定表明,纯化的ADI比活性为80IU/mg。结论：成功构建了ADI的高效表达菌种,建立了目标物质的分离纯化方法。     

重组类胰岛素样生长因子-Ⅰ的纯化与复性

目的 获得高纯度和高活性的胰岛素样生长因子（Insulin-like growth factor, IGF-1）;方法 构建好的BL21大肠杆菌工程菌经IPTG诱导,以融合一段截短型半乳糖苷酶及His-tag形式表达IGF-1融合蛋白(约15,000Da),超声破碎,提取包涵体经镍柱亲和层析后, 用羟氨切割纯化的融合蛋白,纯化后的蛋白质在小分子保护剂及GSH/GSSG的存在下复性。结果 经Ni2＋柱亲和层析, IGF-1纯度达90％以上,复性后得到有较高生物活性的IGF-1。结论 IGF-1发酵及纯化和复性方法的建立为大量生产IGF-1打下了基础。     

Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II

The insulin-like growth factors, IGF-I and IGF-II, are single chain polypeptides, which are structurally related to proinsulin and promote proliferation and differentiation of cells in many vertebrate species. Previous attempts to produce recombinant salmon IGF-II (rsIGF-II) were compromised by low expression levels and co-purification of incorrectly cleaved protein with the authentic recombinant product. In this study, a gene containing the coding region for Atlantic salmon (Salmo salar) IGF-II was cloned into a modified pET32a expression vector and transformed into Escherichia coli BL21 trxB (DE3) cells. Upon growth and induction (with IPTG) of the transformant, recombinant salmon IGF-II (rsIGF-II) was expressed as an insoluble, 28kDa thioredoxin.sIGF-II fusion protein linked by a protease cleavage motif (trx.FAHY.sIGF-II) in inclusion bodies. The inclusion bodies were subsequently solubilized and the fusion protein was purified by Ni-affinity chromatography. Recombinant IGF-II (7.8kDa) was then released from the fusion partner using H64A subtilisin BPN' protease and purified by reversed-phase HPLC. Homogeneity of the final recombinant product was confirmed by N-terminal amino acid sequencing, ion-spray mass spectrometry, SDS-polyacrylamide gel electrophoresis, and analytical reversed-phase HPLC. The biological activity of rsIGF-II was demonstrated in cultured rat L6 myoblasts and was found to be approximately 9- and 5-fold less potent than recombinant human IGF-I and recombinant salmon IGF-I, respectively, a result similar to that demonstrated previously with other recombinant fish IGF-II's in non-homologous cell lines.     

霍乱毒素B亚单位与胰岛素B链融合基因的克隆及原核表达分析

构建了霍乱毒素B亚单位(choleratoxinBsubunit,CTB)与胰岛素(insulin)B链的融合基因CTB-INSB,将该融合基因克隆到大肠杆菌表达载体pET-30a(+)中,获得重组质粒pETCIB;并将该质粒转入大肠杆菌菌株BL21(DE3)中;重组菌株经IPTG诱导后的表达产物经15％SDS-PAGE分析表明可以表达融合蛋白,其分子量约为15.4kDa,且主要以包涵体形式存在,约占全菌蛋白的30％。含CTB-INSB重组蛋白的包涵体经变性和复性后,可在体外自组装成五聚体结构。Westernblotting分析结果显示CTB-INSB可分别被霍乱毒素的抗体和胰岛素的抗体识别,表明该蛋白具有霍乱毒素B亚单位与胰岛素的双重抗原性。同时GM1-ELISA分析结果表明CTB-INSB在体外可与神经节苷脂GM1(monosialoganglioside)特异结合,进一步证实了它能够形成类似CTB五聚体的高级结构,具有生物活性。     

小反刍兽疫病毒N蛋白原核表达条件的优化及反应活性

目的 探讨小反刍兽疫病毒（Peste des Petits Ruminants Virus,PPRV）N蛋白原核表达、条件优化及反应活性。方法 参照GenBank中PPRV(Nigeria/75/1)N基因序列,人工合成该基因,构建重组表达质粒pET-30a(+)-N,转化至BL21(DE3)感受态细胞,IPTG诱导表达,进行SDS-PAGE电泳、Western-blot分析。在不同温度、时间、IPTG浓度条件下诱导表达N蛋白,确定最佳表达条件。结果 PPRV N蛋白（64.4 kD）成功表达,能被羊PPRV免疫血清所识别。N蛋白主要以包涵体存在,其最佳诱导条件：诱导温度28℃、IPTG终浓度2.0 mmol/L、诱导时间16 h。结论 原核表达的PPRV N蛋白具有良好的特异性和反应活性,为单克隆抗体的制备及快速诊断方法的建立提供了技术资料。     

重组PLZF蛋白锌指结构域的表达、提纯和活性分析

PLZF(promyelocytic leukaemia zinc finger protein)是一种重要的转录抑制因子,它由位于N端的BTB结构域和C端的锌指结构域构成。鉴于目前对于锌指结构域的立体结构还不是十分清楚,对其进行了高效表达和提纯。为了表达PLZF蛋白的锌指结构域,在其编码序列的5'端加上起始密码ATG后插入到表达载体PET-11a的多克隆位点。构建好的表达质粒转化到BL21 (DE3)大肠杆菌内并用IPTG诱导表达,发现重组蛋白主要以不溶性的包涵体形式在胞内表达。用含有SDS变性剂的缓冲液溶解包涵体后,采用凝胶过滤方法将重组蛋白纯化到纯度达96%以上。对纯化后的蛋白质用反透析的方法进行复性,然后用DNA结合实验进行活性分析,发现复性后的蛋白质具有特异的DNA结合活性,这为进一步研究PLZF蛋白锌指结构域的立体结构打下了重要基础。     

Production of recombinant mink growth hormone in <Emphasis Type="Italic">E. coli</Emphasis>

Escherichia coli cells expressing mink (Mustela vison) growth hormone were grown in a batch fermentation process. The expression level was estimated to be 27% of the total cellular protein after 3 h of induction with 1 mM isopropyl β-d-thiogalactoside (IPTG). If the expression of mink growth hormone (mGH) was induced with 0.2 mM IPTG, the concentration of target protein was slightly lower and was found to be 23% at the same time after induction. mGH expressed as inclusion bodies was solubilized in 8 M urea and renatured by dilution protocol at a protein concentration of 1.4–2.1 mg/ml in the presence of glutathione pair in a final concentration of 11.3 mM. [GSH]/[GSSG] ratio equal to 2/1 was used. Two-step purification process comprising of ion-exchange chromatography on Q-Sepharose and hydrophobic chromatography on Phenyl-Sepharose was developed. Some 25–30 mg of highly purified and biologically active mGH was obtained from 4 g of biomass. The method presented in this study allows producing large quantities of mGH and considering initiation of scientific investigation on mGH effect on mink in vivo and availability in fur industry.     

人vasorin蛋白的基因克隆、表达及纯化简

目的：克隆、表达人vasorin（VASN）蛋白。方法：利用PCR方法从HepG2细胞的cDNA中扩增获得目的基因,并插入带有6xHis标签的原核高效可溶性表达载体pET28a中,构建重组表达质粒pET28a-VASN,将重组表达质粒转化大肠杆菌BL21（DE3）,经IPTG诱导后目的基因获得表达,对融合目的蛋白进行Ni^2＋金属螯合柱纯化。结果：内切酶鉴定及基因序列测定证实重组表达质粒构建成功;对目的蛋白进行了原核表达,SDS-PAGE显示相对分子质量为61x10^3的特异表达条带;Western印迹证实目的蛋白为VASN,且主要以包涵体形式存在;对经尿素变性的表达产物进行了亲和层析纯化,有利于以后的变性、复性过程。结论：获得了人VASN融合蛋白,为其进一步的生物学功能研究奠定了基础。     

霍乱毒素B亚单位与胰岛素原融合基因的克隆、表达及重组蛋白特性分析

构建了霍乱毒素B亚单位 (CholeratoxinBsubunit,CTB)与胰岛素原 (Proinsulin)的融合基因CTB -PROIN ,将该融合基因克隆到大肠杆菌表达载体pET 30a(+)中 ,获得重组质粒pETCPI,并将该质粒转入大肠杆菌菌株BL21(DE3)中 ;重组菌株经IPTG诱导后 ,其表达产物经过 15 %SDS PAGE分析表明该菌株可以表达融合蛋白 ,其分子量约为 21.6kD ,且主要以包涵体形式存在 ,约占全菌蛋白的 25%。含CTB-PROIN重组蛋白的包涵体经过变性和复性后 ,CTB-PROIN可以在体外自组装成五聚体结构。Westernblotting分析结果表明重组CTB PROIN蛋白可分别被霍乱毒素的抗体和胰岛素的抗体识别 ,说明该蛋白具有霍乱毒素和胰岛素的双重抗原性。同时在体外 ,CTB PROIN蛋白可与神经节苷脂GM1(monosialoganglioside)特异性结合 ,表明了该融合蛋白在体外具有生物活性。这些研究结果为利用原核生物表达系统研制廉价、高效的I型糖尿病口服疫苗奠定了基础。     

High-level expression, purification and pro-apoptosis activity of HIV-TAT-survivin (T34A) mutant to cancer cells in vitro

Survivin, a novel member of the IAP family, was observed to express in the most common human cancers. Anti-cancer therapy targeting survivin had drawn considerable attention. This study focused on high-level expression of recombinant protein TAT-survivin (T34A) mutant in E. coli, purification and bioactivity of pro-apoptosis to various cell lines in vitro. The cDNA encoding survivin was cloned by RT-PCR from breast cancer cell lines B-cap37. After PCR site-directed mutagenesis and construction of expression vector pRSET-B-TAT-survivin (T34A), targeted TAT-survivin (T34A) protein was expressed highly in E. coli BL21 (DE3) by 0.5mM IPTG induction and its yield could reach 650 mg/l in fermentation culture. The fusion protein in a form of inclusion body was then solubilized, refolded and purified to a purity of 98% by cation exchange chromatography and size-exclusion chromatography. Four hundred and eighty milligrams protein of interest was obtained in per liter fermentation culture. This showed that the efficient procedures of large-scale expression and purification were successful for the mass production of the recombinant protein. Pro-apoptosis effects of target protein on four cancer cell lines and one normal cell line from human were confirmed by the change of morphology, and pro-apoptosis activity was evaluated by MTT, fluorescent staining of nuclei and flow cytometry assay. Results indicated that B-cap37 and SW1990 were very sensitive to TAT-survivin (T34A) protein. This finding revealed the recombinant protein was promising as an anti-cancer drug.     

嗜热古菌S-腺苷甲硫氨酸合成酶的序列分析、基因克隆与异源表达

将Methanopyrus sp.SNP6进行功能基因组测序,获得S-腺苷甲硫氨酸合成酶基因(sam)序列,并进行序列分析。将sam基因扩增后连接至表达质粒p ET-28b(+),转化E.coli BL21(DE3),获得重组菌后进行诱导表达。生物信息学分析表明,sam基因编码蛋白的理论分子量为44 086.4Da,三级结构为同源四聚体,与其他相关古菌来源的S-腺苷甲硫氨酸合成酶的蛋白序列较保守。实验结果显示,构建的重组表达质粒p ET28b(+)-sam可在E.coli BL21(DE3)宿主菌中高水平表达。重组蛋白的分子量与预期值基本一致,部分为胞内可溶性表达,另一部分以包涵体形式存在。本研究首次实现了Methanopyrus sp.SNP6菌株S-腺苷甲硫氨酸合成酶的异源表达,为后期的蛋白纯化、酶学性质研究和酶促转化法生产S-腺苷甲硫氨酸奠定了理论基础。